The science and instrumentation of spectroscopy as developed over the last century has become increasingly expanded and specialized as the various methods and applications of analysis came into existence. Today, spectroscopy has been divided into individual and distinctly different methods and instrumentation systems for: ultraviolet and visible spectrophotometry; fluorescence and phosphorescence spectrometry; atomic emission spectroscopy; infrared spectrophotometry; raman spectroscopy; nuclear magnetic resonance spectroscopy; electron spin resonance spectroscopy; refractometry and interferometry; and various others. Of these, the optical sensors and optical sensing detection systems utilizing the ultraviolet and visible absorption methods and the fluorescence and phosphorescence excitation and emission systems are perhaps the best known and commonly utilized.
In particular, the use of optical fibers and optical fiber strands in combination with light energy absorbing dyes for medical, environmental, and chemical analytical determinations has undergone rapid development especially within the last decade. The use of optical fibers for such purposes and techniques generally is described by the following publications: Milanovich et al., "Novel Optical Fiber Techniques for Medical Application," Proceedings of the SPIE 28th Annual International Technical Symposium on Optics and Electro-Optics, Volume 494, 1980; Seitz, W. R., "Chemical Sensors Based on Immobilized Indicators and Fiber Optics," in C.R.C. Critical Reviews in Analytical Chemistry, Vol. 19, 1988, pp 135-173; Wolfbeis, O. S., "Fiber Optical Fluorosensors in Analytical Chemistry," in Molecular Luminescence Spectroscopy, Methods and Applications (S. G. Schulman, editor), Wiley & Sons, New York (1988); Angel, S. M., Spectroscopy 2(4):38 (1987); Walt et al., "Chemical Sensors and Microinstrumentation," ACS Symposium Series, volume 403, 1989, p 252; and Wolfbeis, CRC Press, 1991.
The optical fiber strands employed for analytical determinations typically are glass or plastic extended rods having a small cross-sectional diameter. When light energy is projected into one end of the fiber strand (conventionally termed the "proximal end"), the angles at which the various light energy rays strike the surface are greater than the critical angle; and such rays are "piped" through the strand's length by successive internal reflections and eventually exit from the opposite end of the strand (conventionally termed the "distal end"). Typically, bundles of these strands are used collectively as optical fibers in a variety of different applications.
For making an optical fiber into a sensor, one or more light energy absorbing dyes are attached to the distal end of the optical fiber. The sensor can then be used for both in-vitro and/or in-vivo applications. As used herein, light energy is photoenergy and is defined as electromagnetic radiation of any wavelength. Accordingly, the terms "light energy" and "photoenergy" include infrared, visible, and ultraviolet wavelengths conventionally employed in most optical instruments and apparatus; the term also includes the other spectral regions of x-ray and microwave wavelengths (although these are generally not used in conjunction with optical fibers).
Typically, light from an appropriate energy source is used to illuminate what is chosen to be the proximal end of an optical fiber or a fiber bundle. The light propagates along the length of the optical fiber; and a portion of this propagated light energy exits the distal end of the optical fiber and is absorbed by one or more light energy absorbing dyes. As conventionally known, the light energy absorbing dye may or may not be immobilized; may or may not be directly attached to the optical fiber itself; may or may not be suspended in a fluid sample containing one or more analytes of interest to be detected; and may or may not be retainable for subsequent use in a second optical determination.
Once the light energy has been absorbed by the dye, some light energy of varying wavelength and intensity typically returns through the distal end of the optical fiber and is then conveyed through either the same fiber or a collection fiber or fibers to a detection system where the emerging light energy is observed and measured. The interactions between the incoming light energy conveyed by the optical fiber and the properties of the light absorbing dye--both in the presence of a fluid sample containing one or more analytes of interest and in the absence of any analytes whatsoever--provide an optical basis for both qualitative and quantitative spectral determinations. Merely illustrating the use of some presently known optical fiber sensors in a variety of different conditions, apparatus, dyes, and applications are U.S. Pat. Nos. 4,822,746; 4,144,452; 4,495,293; and Re. 31,879.
Moreover, in view of the microcircuitry and enhanced television technology presently available, a variety of light image processing and analytical system have now come into existence in order to enhance, analyze, and mathematically process the light energies introduced to and emerging from the absorbing dyes in such optical analytical techniques. Typically, these systems provide components for image capture; data acquisition; data processing and analysis; and visual presentation to the user. Commercially available systems include the QX-7 image processing and analysis system sold by Quantex, Inc. (Sunnydale, Calif.); and the IM Spectrofluorescence imaging system offered by SPEX Industries, Inc. (Edison, N.J.). Each of these systems may be combined with microscopes, cameras, and/or television monitors for automatic processing of all light energy determinations.
Of the many different classes of light absorbing dyes which may be employed with single optical fiber strands and with bundles of optical fibers for different analytical purposes are those compositions which emit light energy after first absorbing energy and are termed "fluorophores"; and those compositions which absorb light energy and internally convert the absorbed light energy into heat or kinetic energy rather than emit it as light and are termed "chromophores" or "absorbers". Fluorophores and fluorescent detection methods employing optical fibers are recognized as being markedly different and distinguishable from light energy absorbance and absorption spectroscopy.
Fluorescence is a physical phenomenon based upon the ability of some molecules to absorb light energy (photons) at specified wavelengths and then emit light energy of a longer wavelength and at a lower energy. Such emissions are called fluorescence if the emission is relatively long-lived, typically 10.sup.-11 to 10.sup.-7 seconds. Substances able to fluoresce share and display a number of common characteristics: they absorb light energy at one wavelength or frequency; reach an excited energy state; and subsequently emit light at another light frequency and energy level. The absorption and fluorescence emission spectra are thus individual for each fluorophore; and are often graphically represented as two separate curves which are slightly overlapping.
All fluorophores demonstrate the Stokes' shift--that is, the emitted light is always at a longer wavelength (and at a lower energy level) relative to the wavelength (and energy level) of the exciting light absorbed by the substance. Moreover, the same fluorescence emission spectrum is generally observed irrespective of the wavelength of the exciting light and, accordingly, the wavelength and energy of the exciting light may be varied within limits; but the light emitted by the fluorophore will always provide the same emission spectrum as emerging light. Finally, fluorescence may be measured as the quantum yield of light emitted. The fluorescence quantum yield is the ratio of the number of photons emitted in comparison to the number of photons initially absorbed by the fluorophore. For more detailed information regarding each of these characteristics, the following references are recommended: Lakowicz, J. R., Principles of Fluorescence Spectroscopy, Plenum Press, New York, 1983; Freifelder, D., Physical Biochemistry, second edition, W. H. Freeman and Company, New York, 1982; "Molecular Luminescence Spectroscopy Methods and Applications: Part I" (S. G. Schulman, editor) in Chemical Analysis, vol. 77, Wiley & Sons, Inc., 1985; The Theory of Luminescence, Stepanov and Gribkovskii, Iliffe Books, Ltd., London, 1968.
In comparison, substances which absorb light energy and do not fluoresce usually convert the light energy into heat or kinetic energy. The ability to internally convert the absorbed light energy identifies the dye as a "chromophore." Dyes which absorb light energy as chromophores do so at individual wavelengths of energy and are characterized by a distinctive molar absorption coefficient for light energy at that wavelength. Chemical analyses employing fiber optic strands and absorption spectroscopy using visible and ultraviolet light wavelengths in combination with the absorption coefficient allow for the determination of concentration for specific analytes of interest by spectral measurement. The most common use of absorbance measurement via optical fibers is to determine concentration which is calculated in accordance with Beers' law; accordingly, at a single absorbance wavelength, the greater the quantity of the composition which absorbs light energy at a given photowavelength, the greater the optical density for the sample. In this way, the total quantity of light absorbed directly correlates with the quantity of the composition in the sample.
Many of the recent improvements employing optical fiber sensors in both qualitative and quantitative analytical determinations concern the desirability of depositing and/or immobilizing various light absorbing dyes at the distal end of the optical fiber using a given technique or apparatus. In this manner, a variety of different optical fiber chemical sensors and methods have been reported for specific analytical determinations and applications such as pH measurement, oxygen detection, and carbon dioxide analyses. These developments are represented and exemplified by the following publications: Freeman et al., Anal. Chem. 53:98 (1983); Lippitsch et al., Anal. Chem. Acta. 205:1, (1988); Wolfbeis et al., Anal. Chem. 60:2028 (1988); Jordan et al., Anal. Chem. 59:437 (1987); Lubbers et al., Sens. Actuators, 1983; Munkholm et al., Talanta 35:109 (1988); Munkholm et al., Anal. Chem. 58:1427 (1986); Seitz, W. R., Anal. Chem. 56:116A-34A (1984); Peterson et al., Anal. Chem. 52:864 (1980); Saari et al., Anal. Chem. 54:821 (1982); Saari et al., Anal. Chem. 55:667 (1983); Zhujun et al., Anal. Chem. Acta. 160:47 (1984); and Schwab et al., Anal. Chem. 56:2199 (1984).
Concurrent with developments in fiber optic technology have been the dramatic and devastating changes in our environment. Over the last several decades there has been increasing awareness and concern over organic contamination from hazardous waste sites and underground storage tanks. This contamination threatens the quality of groundwater at aquifers, thereby polluting the only drinking water source in many communities. These concerns have generated a massive effort of sampling and analysis at an ever-increasing number of monitoring wells. Existing monitoring technology [as described in Koehn, J. W. and G. H. Stanko, Jr., Environ. Sci. Technol. 22:1262-1263 (1988)] relies typically on expensive, labor-intensive, discrete sample methods that introduce uncertainties in the sampling and handling procedures. Often there is a long delay between sample collection and communication of results caused by the inability of conventional methods to provide in situ real-time monitoring. Moreover, extensive documentation is required due to chain-of-custody concerns. The application and generation of a low-cost reliable monitoring system employing fiber optic sensors and fiberoptic detection apparatus would reduce the need for frequent samples and provide timely continuous information of water quality.